1. Field of the Invention
Provided are formulations and methods of producing active cytofectin:polynucleotide transfection complexes from a collection of cytofectins (cytofectins are defined as chemical species that are cationic transfection amphiphiles) or cationic lipids that bind and transport polynucleotides through membrane barriers. More specifically, structural features in cytofectins that improve transfection are selected, cytofectin counterions that improve transfection are chosen, and heating/sonication conditions for the formation of transfectin:polynucleotide complexes are maximized.
2. Description of the Background Art
Cellular transfection strategies for gene therapy and similar goals have been designed and performed, but many of these procedures involve recombinant virus vectors and various problems exist with these viral gene transfer systems. Even generally advantageous adenovirus techniques encounter difficulties since most humans have antibodies to many of the adenovirus serogroups, including those that have been chosen as vectors. Wild type adenoviral superinfection of an adenoviral vector treated patient may result in propagating the recombinant vector as a defective viral particle, with the ability to infect many unintended individuals (if chosen to have a rare serogroup). The chance of adenoviral contamination is quite low but not impossible. The safety of using these genetic materials in humans remains unclear and thus hazardous.
Unfortunately, the potential of gene transfer-based research to improve human health will be restricted unless improved methods are developed for in vivo delivery of foreign genetic material into cells and tissues. Currently used viral and non-viral transfection reagents have been compromised by one or more problems pertaining to: 1) associated health risks, 2) immunological complications, 3) inefficient in vivo transfection efficiency, and 4) direct cytotoxicity. The development of safe and effective polynucleotide-based medicines will require improved solutions which address these problems. Therefore, safe, non-viral vector methods for transfection or gene therapy are essential.
Cationic amphiphiles are currently regarded as an alternative to viral vector technology for in vivo polynucleotide delivery. Cationic lipid-based reagents avoid many of the health and immunological concerns associated with viral vectors. In a practical sense, cationic amphiphile-based delivery agents are relatively simple to use, and offer unparalleled flexibility in the nature of the material that can be delivered. Typically, cationic lipid complexes are prepared by mixing the cationic lipid (cytofectin) with the desired DNA (1), RNA (2), antisense oligomer (3), or protein (4) to yield active particles; in contrast to the laborious recombinant DNA and cell culture manipulations which are typically required to produce virus-derived delivery agents.
A few such lipid delivery systems for transporting DNA, proteins, and other chemical materials across membrane boundaries have been synthesized by research groups and business entities. Most of the synthesis schemes are relatively complex and generate lipid based delivery systems having only limited transfection abilities. A need exists in the field of gene therapy for cationic lipid species that have a high biopolymer transport efficiency. It has been known for some time that a very limited number of certain quaternary ammonium derivatized (cationic) liposomes spontaneously associate with DNA, fuse with cell membranes, and deliver the DNA into the cytoplasm (as noted above, these species have been termed "cytofectins"). LIPOFECTIN.TM. represents a first generation of cationic liposome formulation development. LIPOFECTIN.TM. is composed of a 1:1 formulation of the quaternary ammonium containing compound DOTMA and dioleoylphosphatidylethanolamine sonicated into small unilamellar vesicles in water. Problems associated with LIPOCFECTIN.TM. include non-metabolizable ether bonds, inhibition of protein kinase C activity, and direct cytotoxicity. In response to these problems, a number of other related compounds have been developed. The monoammonium compounds of the subject invention improve upon the capabilities of existing cationic liposomes and serve as a very efficient delivery system for biologically active chemicals.
Since the original report (Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M. and Danielsen, M. 1987. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84(21): 7413-7, which is herein incorporated by reference, as are all cited references in this disclosure) that liposomes comprised of equal amounts of the cytofectin DOTMA (N-1-(2,3-dioleyloxy)propyl!-N,N,N-trimethylammonium chloride) and neutral lipid DOPE (dioleoyl phosphotidylethanolamine) spontaneously associate with DNA to form efficient transfection complexes, the technology has advanced incrementally. There have been few cytofectins developed which have improved upon the in vivo activity of the prototypic agent DOTMA. This lack of progress may reflect funding priorities which have focused on the application of cationic lipid technology to biologic problems, rather than research focusing on principles which effect cytofectin-mediated gene delivery. Specifically, studies focused on the mechanism(s) involved in cytofectin actions, barriers to cytofectin-mediated in vivo gene delivery, and clarification of cytofectin structure/activity relationships would facilitate the development of improved cationic lipid-based delivery reagents. While research into the mechanism responsible for cationic amphiphile-mediated gene delivery is ongoing in a number of laboratories (Sternberg, B., Sorgi, F. L. and Huang, L. 1994. New structures in complex formation between DNA and cationic liposomes visualized by freeze-fracture electron microscopy. FEBS. Lett. 356(2-3): 361-6, Wrobel, I. and Collins, D. 1995. Fusion of cationic liposomes with mammalian cells occurs after endocytosis. Biochim. Biophys. Acta 1235(2): 296-304, and Zabner, J., Fasbender, A. J., Moninger, T., Poellinger, K. A. and Welsh, M. J. 1995. Cellular and molecular barriers to gene transfer by a cationic lipid. J. Biol. Chem. 270(32): 18997-9007), even the most basic aspects of the mechanism of action of cytofectins (the relative contributions of direct cytoplasmic membrane fusion and endocytosis) remain unresolved.
Currently, several cationic amphiphile preparations are commercially available, and new analogs have been published. However, these agents are frequently reported without comparison to existing compounds, and therefore it is difficult to derive insights into the relationship of structural motifs to polynucleotide transfection. This difficulty has been exacerbated by the variability in: 1) transfected cell types exploited in the initial report, 2) reporter genes used to characterize transfection, 3) methods for reporting biologic response (typically reporter protein expression) and 4) specific expression vector design. In addition, there have been few reports which describe the effects of alternative formulation methods. Paradoxically, the relative lack of such fundamental information implies that significant improvements in cytofectin-mediated gene transfer technology may be achieved by further systematic study.
As indicated above, various cationic lipids have been synthesized in previous references. In the realm of patents, for example, U.S. Pat. No. 4,812,449 discloses in situ active compound assembly of biologically active agents at target locations in preference to surroundings which are desired to be unaffected. Several charged and uncharged amine derivatives are described.
Introduced in U.S. Pat. No. 5,171,678 are lipopolyamines and their use for transfecting eukaryotic cells. A polynucleotide is mixed with the subject lipopolyamine and contacted with the cells to be treated.
U.S. Pat. Nos. 5,186,923 and 5,277,897 relate an enhancement of cellular accumulation of lipophilic cationic organometallic compounds by reduction of the intramembrane potential. Technetium containing compounds are disclosed.
Lipophilic cationic compounds are presented in U.S. Pat. No. 5,208,036. Asymmetrical amine compounds are synthesized and employed in a method for DNA transfection. The amines are quaternized by two hydrogens or alkyl, aryl, aralkyl, quinuclidino, piperidino, pyrrolidino, or morpholine groups, unlike the present invention.
U.S. Pat. No. 5,264,618 discloses cationic lipids for intracellular delivery of biologically active molecules. Asymmetric ammonium containing cationic lipids are presented for transporting molecules into membrane enclosed systems. The amines are quaternized by two hydrogens or alkyl groups, unlike the present invention.
Transfection of nucleic acids into animal cells via a neutral lipid and a cationic lipid is revealed in U.S. Pat. No. 5,279,833. Liposomes with nucleic acid transfection activity are formed from the neutral lipid and the ammonium salt containing cationic lipid.
U.S. Pat. No. 5,334,761 describes other amine containing cationic lipids. Cationic lipids are utilized to form aggregates for delivery of macromolecules and other compounds into cells. The amines are quaternized by two hydrogens or unbranched alkyl groups, unlike the present invention.
In the PCT publication of PCT/US94/13362 a heterocyclic diamine is disclosed. A symmetrical quaternary diamine having lipid tails is related for forming liposomes.
The foregoing patents and publication reflect the state of the art of which the applicants are aware and are tendered with the view toward discharging applicants' acknowledged duty of candor in disclosing information which may be pertinent in the examination of this application. It is respectfully submitted, however, that none of these patents teach or render obvious, singly or when considered in combination, applicants' claimed invention.